Procedures for counting or quantifying cells in milk samples fall into one of two categories. In the first category, bovine somatic cells in raw milk samples are counted by various means to identify milk producing animals that may have bovine mastitis, an undesirable condition which limits the quality and quantity of milk production in infected animals. Mastitis testing procedures include direct cell counting using automated instruments and bioluminescent somatic cell ATP determinations following cell lysis with agents such as detergents that release cellular adenosine triphosphate (ATP). The number of somatic cells originally present in the sample is estimated from the measurement of the ATP released.
In the second category, cells of simple, usually unicellular microorganisms, such as fungi and bacteria, referred to hereinafter collectively as "microorganism cells," are counted in milk samples using various counting procedures. These procedures are generally used in the assessment of milk quality, particularly to screen out grossly contaminated milk samples.
Of the procedures used for microorganism detection, the Breed Smear (Breed, R. S., Zbl. Bakt. Ilte. Abr. 30:337 (1911)) is generally the quickest method. In this technique, a milk sample is smeared onto a slide, dried, stained, and the bacteria are counted using microscopic examination. The drawbacks of this procedure are that both viable and non-viable organisms are counted, and if milk samples contain fewer than 10.sup.5 organisms/ml, many fields in the microscope must be counted to obtain statistically valid results. Microscopic evaluations are tedious and lead to operator fatigue.
The most widely utilized milk microorganism detection method is the standard plate method, which utilizes direct colony counting after plating in or on a growth medium. Standard methods (Standard Methods for the Examination of Dairy Products, 15th Ed., 1985, Richardson, G. H., Ed., American Public Health Org. Washington, D.C.) have been developed for milk samples, and many laboratories evaluate milk samples using either manual or automated plating procedures. While these methods have been utilized worldwide, there are certain disadvantages in using them. First, in the manual plating methods, two or more dilutions of the milk sample must be plated so that statistically significant plate numbers may be obtained. Second, plates for both the manual and automated plating procedures are usually incubated at elevated temperatures (e.g., in the U.S., 35.degree. C.; in Japan, 32.degree. C.); and, at these temperatures, the growth of psychrophillic organisms is repressed, yielding artificially low plate counting numbers. Finally, incubation periods of about 48 hours are required before bacterial plates can be counted and still longer plating periods are necessary for fungi. During this incubation period, bacterial numbers are increasing in the bulk milk from which the sample was taken, so that the result obtained is an underestimate of the actual number of colony forming organisms in the milk after the test. The delay in processing the raw milk to accommodate this incubation period by itself lowers the quality of the raw milk, and can contribute to shorter shelf lifetimes of the final product.
Plating or colony counting methods can be either manual or automated. Manual methods include the plate loop method (Thompson, D. I., Journal of Milk and Food Technology 30, 273 (1967)) and the Standard Plate Count, supra.
Semi-automated or automated colony counting methods include the Spiral Plating Method (Gilchrist, J. E., Appl. Micro. 25, 244 (1973)) and methods carried out by electronic colony counters (Fleming, M. G. Ir. J. Agric. Res. 14, 21 (1975)). The Direct Epi-Fluorescent Technique (DEFT) is a fluorescence-labeling technique which can be performed manually or with automatic instrumentation. Other techniques include impedance measurements after growth of milk organisms and radiometric procedures utilizing radioactive glucose.
Another approach to microbe evaluation has been the utilization of the bioluminescent measurement of ATP from living cells in milk using firefly luciferase (Lumac.RTM. bv, The Netherlands). In this scheme somatic bovine cells in raw milk are lysed with a detergent, which releases somatic cell ATP. This ATP from bovine cells, and any other non-microbial milk ATP, is degraded with an ATPase, usually potato apyrase. Finally, a bacterial lysing agent is added and bacterial ATP is measured in a bioluminescence assay using firefly luciferase. While much data has accumulated in the literature on these methods, the sensitivity has been inadequate for routine milk testing due to milk and extractant inhibition of the luciferase reaction, incomplete removal of non-bacterial ATP, deleterious effects of apyrase on bacterial ATP detection (Theron, D. N., J. Food Prod. 49:4-7 (1986)) both before and after bacterial cell lysis, and inefficient extraction of bacterial ATP. Literature data (Webster et al., J. Food Prod. 51:949-954 (1988)) for this type of assay suggest a cell sensitivity of approximately 1.times.10.sup.6 cells/ml, which cannot be utilized in the United States where a cutoff for acceptance of 1.times.10.sup.5 cells/ml for Grade A raw milk is required.
A logical improvement of this method that has been tried is concentrating the milk bacteria prior to the ATP assay. Various techniques have appeared in the literature which make use of cell filtration concentration (Peterkin and Sharpe, Appl. Environ. Microbiol., 39:1138-1143 (1980)), although these techniques are quite cumbersome, slow, and still exhibit most of the technical problems discussed above.
Lin et al., J. Dairy Science 72:351-359 (1989), describe a method of separating cells from various defined yogurt cultures grown in skim milk. In the method, 1% EDTA at pH 12 is added to the yogurt culture to bring the EDTA concentration to .gtoreq.20 mg/ml of culture and the culture is centrifuged to separate a cell pellet. The cells of the pellet are then analyzed for .beta.-galactosidase activity after being washed with phosphate buffer and disrupted by sonication. There is no teaching in Lin et al. that the enzymatic activity which they measure is correlated to bacterial contamination of milk. Lin et al. do not teach a method of separating cells from, or a method for assessing bacterial contamination of, milk or any other food or material of biological origin.
While all of the techniques mentioned above have demonstrated some measure of success, none has proven to be inexpensive, simple, accurate, fast and sensitive enough to provide the milk industry with a type of test which can be used satisfactorily for routine testing.
Methods of assaying bacterial cultures grown from food (including milk and meat) samples for Salmonella contamination using an immunoassay technique, employing antibodies to an antigen common to Salmonella spp., and a nucleic acid probe hybridization technique, employing DNA probes for Salmonella DNA, are available. These methods, however, require cumbersome and time-consuming growth of bacterial cultures, from the food material being analyzed, before the assays for Salmonella can be carried out.
The need for such culturing prior to application of nucleic acid probe hybridization techniques to detect contamination, by undesirable microorganisms, of food materials, including milk, meat (e.g., chicken, turkey, beef, pork, horse, goat, whale and the like), eggs, fish, mussels, molluscs, crustaceans, vegetables, fruits, grains, and the like can be avoided, or the time for culture significantly reduced, if nucleic acid amplification techniques, such as the well known polymerase chain reaction (PCR) technique, are employed to increase the concentration of nucleic acid segments, characteristic of microorganism contaminants, to levels that are readily detectable by nucleic acid probe hybridization or nucleic acid staining methods. However, target nucleic acid amplification techniques have not been successfully applied for this purpose with cultures or extracts of specimens of food materials, or other materials of biological origin, such as blood, urine or stool, unless, prior to application of processes for amplification, microorganisms from the specimens have been subjected to cumbersome, costly, and otherwise undesirable, special treatments, such as with proteolytic enzymes, high concentrations of guanidinium salts, and detergent, or boiling, and the DNA from the microorganisms has been processed by similarly undesirable procedures such as ethanol-precipitation or chromatographic separation with or without phenol/chloroform extraction. These treatments have been regarded as necessary to separate nucleic acid, to be subjected to amplification, from contaminants, that interfere with the enzymatic reactions necessary for the amplification and that are thought to be provided by the microorganisms themselves or otherwise provided from the specimens to the microorganisms separated therefrom. See, e.g., Hill et al., Appl. Environ. Microbiol. 57:707-711 (1991); Keasler and Hill, Abstracts of the 91st General Meeting of the American Society for Microbiology, Abstract No. P-12, p. 269 (1991); Olive, J. Clin. Microbiol. 27:261-265 (1989).